Yeast-two-hybrid assay

The Y2H screen was performed using the Matchmaker Gold Yeast-Two-Hybrid System and the Mouse Universal Normalized cDNA library (Clontech, Mountain View, CA). The screen was performed following the recommendations of the manufacturer. The GAL4 DNA-BD/bait construct was prepared by ligating a PCR fragment containing the AnkB death domain (aa1485-1563, accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_020977.3","term_id":"188595666","term_text":"NM_020977.3"}}NM_020977.3) into the pGBKT7 vector (Clontech). The yeast strain Y2HGold was maintained on YPD agar plates on SD –TRP (tryptophan) plates when transfected with the GAL4 DNA-BD/AnkB DD bait construct. To confirm the expression of the bait and prey plasmids, cells were grown on SD-Leu/Trp plates. The library was transformed into the AH 109 yeast strain (Takara Bio Inc., Mountain View, CA). The Y2H screen was performed under high-stringency growth conditions as recommended by the manufacturer.

Yeast cells coexpressing either AnkB DD or AnkG DD (baits) together with either the C-terminal sequence of RabGAP1L cloned in pGADT7, or the empty pGADT7vector (prey) were grown on plates lacking leucine, tryptophan, histidine, and adenine (-Leu, -Trp, -His, and -Ade) and supplemented with 125 ng/ml Aureobasidin A. Same conditions were used to assess interactions between AnkB DD mutant baits and the RabGAP1L C-terminal domain.